**4. Fluorescence**

114 Aflatoxins – Detection, Measurement and Control

be combined with the specification and sensitivity for an enzymatic reaction (Girelli & Mattei, 2005). Derivatization with a fluorophore enhances the natural fluorescence of aflatoxins and improves detectability. The pre-column approach uses the formation of the corresponding hemiacetals using trifluoroacetic acid (TFA), while the post-column one utilizes either bromination by an electrochemical cellor in addition of bromide, or pyridinium hydrobromide perbromide, for the mobile phase and the formation of an iodine

Even though the optical devices have dominated the traditional methods for HPLC, the present trend is to use mass detectors in the different HPLC types and configurations. This is because of the universal, selective and sensitive detection they provide (Alcaide-Molina,

There are several techniques that use chromatography for aflatoxin analysis in food (principally in milk, cheese, corn, peanuts, nuts). Commonly the quantification of the aflatoxins is made by a fluorescence detector that takes advantage of fluorescence properties of aflatoxins under determined wavelength. As a result, researchers have been focused on improving these fluorescence properties to develop more sensitive methods than the commonly used so far. Currently techniques such as pre-column derivatization and postcolumn derivatization are commonly used to improve aflatoxins fluorescence properties. They also have a clean-up stage to obtain a more pure sample, permiting a better quantification. Some of the common methods used in the clean-up stage are:

HPLC is a method for detection of aflatoxins which often is enhanced by other techniques, resulting on alternative chromatographic methods. Accomplishing techniques related to electrokinetics are: Micellar electrokinetic chromatography (MEKC), reversed flow micellar electrokinetic chromatography (RFMEKC), and capillary electrokinetic chromatography (CEKC) with multiphoton excited fluorescence (MPE) detection, among others (Gilbert &

Electrokinetics consists on an interfacial double layer of charges effect in heterogeneous fluids (Rathore and Guttman, 2003). Such effect generates the motion of the fluid due to an external force. This external force may be of different natures, but it is called electrophoresis when the force is an electric field; and capillary osmosis when the force is a chemical

Capillary electrophoresis is a technique that although not been widely available as an alternative in many laboratories which routinely conduct HPLC, it has the advantage that it avoids the use of organic solvents. aflatoxin B1 can be determined by capillary electrophoresis (CEKC) with laser-induced fluorescence (LIF) detection (Maragos & Greer, 1997) after a clean-up process comparable to that required for HPLC, and with a very similar sensitivity to it. Besides, Electrophoresis does not require derivatization of aflatoxins, being that an advantage over HPLC. Sensitivity on CEKC can be further improved by using multiphoton excitation. Detection at levels 104 better than previously achieved by capillary separation in less than 90 seconds can be reached, which demonstrates the potential of this

Micellar electrokinetic chromatography (MEKC) is conducted in polyacrylamide-coated capillaries under almost complete suppression of electroosmotic flow (Janini et al., 1996).

potential gradient and the motion of liquid happens in a porous body.

immunoaffinity column and solid phase extraction.

derivative.

**3.4 Electrokinetics** 

Vargas, 2003).

technique (Wei et al., 2000).

2009).

All the aflatoxins have a maximum absorption around 360 nm (Akbas and Ozdemir, 2006). Letters 'B' and 'G' of the aflatoxins refer to its blue (425nm) and green-blue (450nm) fluorescence colours produced by these compounds under Ultra Violet (UV) light. AFB1 is the most common aflatoxin; it is followed by the AFB2. AFG is fairly rare. The fluorescence emission of the G toxin is more than 10 times greater than that for the B toxin (Alcaide-Molina et al., 2009).

Different techniques for detection of AFs related to fluorescence are exposed bellow.

#### **4.1 Black light test**

The black light test is a method which correctly identifies negative AFs samples with minimum expenditure of time and money. It consists on the illumination of the sample with a UV lamp. Tests should be made in a darkened area for best contrast. Fluorescence may be bright or dim, depending on the amount of fluorescing agent present. Polished metal surfaces reflect blue light, thus, users must be careful distinguishing fluorescence from such reflection. It is highly recommended to use safety goggles when working with the black light test. These goggles eliminate blue haze resulting from eye fluorescence caused by reflected longwave UV radiation.

However, fluorescence does not happen exclusively when aflatoxins are present. There are other substances in food that fluoresce under long wave UV radiation. Fungi as *Aspergillus niger*, various *Penicillium* species, *Aspergillus repens* and other species do not produce aflatoxins, but may produce fluorescent harmless metabolites. Then, it can be said that fluorescence is not a specific indication of the presence of aflatoxins, although it may indicate that conditions have been favourable for growth of toxic molds (B-100 Series Ultraviolet Lamps, UVP).

Furthermore, fluorescence is not stable. It disappears in 4 to 6 weeks of continuous exposure to visible or UV radiation although the toxin remains. Therefore, fresh samples must be taken. Hence, the reliability of the method depends on the size of the sample taken for analysis and how it is taken. A sample must be large enough to be representative of the entire lot and must be taken from all parts of the lot (B-100 Series Ultraviolet Lamps, UVP).

The black light test is commonly applied on animal feed. However, it is only a preliminary confirmatory test; it does not give a quantitative indication. Thus confirmatory and quantitative measurements are needed to be applied to those samples that reacted positively to the black light test. Non-fluorescing samples need not be subjected to this. A quantitative screening test which commonly follows the black light test is small chromatographic column (mini-column) (B-100 Series Ultraviolet Lamps, UVP). After the quantitative test a judgment can be made as to whether or not accept a lot.

#### **4.2 Laser-Induced Fluorescence (LIF) screening method**

LIF detection technique was pioneered by Yeung (Novotny & Ishii, 1985). This screening method consists on a mobile phase which contains an eluted sample of aflatoxins. Such

Methods for Detection and Quantification of Aflatoxins 117

Fluorescence detection and electrochemical detection are the two sensitive detection means most commonly used for quantitative studies in HPLC. This happens because the sensitivity levels of those hybrid techniques are much better than the ones observed with conventional fluorescence. It has been demonstrated the usefulness of LIF for sensitive detection in HPLC and micro high-performance liquid chromatography (µHPLC) in sensing very low concentrations of substances that can be excited in the near-UV range (325 nm) after labelling at nanomolar concentrations (Folestad et al., 1985; Diebold et al., 1979). Thus, LIF-HPLC method has become very popular and an essential detection technique in capillary electrophoresis (CE). Its sensitivity has been increased by the use of photoactivation devices (Reif & Metzger, 1995). Its popularity is due to its capability to detect substances at lower ranges than the micromolar (Bayle et al., 2004). For more information about HPLC refer to

It has been said that in LIF detection, the number of molecules that are photo-degraded is inversely proportional to the velocity of the fluorophore in front of the laser beam. On the other side, the sensitivity of detection in HPLC depends on the inner diameter of the capillary connected to the output of the column. Therefore, at a constant flow-rate, the sensitivity depends on the velocity of the fluorophore in front of the laser beam of the LIF, and the solid angle of fluorescence collection by the optical arrangement (Simeon et al., 1999). As a result, the union of LIF and HPLC offers a good compromise between sensitivity

In flow injection experiments with LIF-HPLC systems, at a given diameter, the detector signal will increase when increasing flow-rates if photochemical degradation is a limiting factor (Simeon et al., 2001). Conversely, if the flow-rate is fixed, an increase in diameter is expected to lead to a quadratic increase in the detector volume, generating also a quadratic increase in the number of detectable molecules. Then can be said that if a larger volume is irradiated at a larger capillary diameter, the efficiency of fluorescence collection is less

Since Fluorescence systems have a wide sensitivity, they are a useful tool to measure AFM1 in milk, which legal limit is very low (about 50 ppt). These systems are suitable for preliminary screening at the earlier stages of the industrial process, and make it possible to discard contaminated milk stocks before their inclusion in the production chain (Cucci et al., 2007). PMTs are highly sensitive photomultipliers based flow through detection system suited for ultra low fluorescence, chemiluminescence or bioluminescence measurements (PMT-FL, FIAlab Instruments). Their photon counting photon counting sensor has a bluegreen (280–630 nm) spectral response with a peak of quantum efficiency at 400 nm and ultra-low dark counts. The high sensitivity of these devices reaches parts per trillion, permitting measurements of extremely low fluorescence signals. These devices may work with an internal excitation lamp, a LED source or an SMA terminated fiber optic cable for use with an external lamp. They also count with removable emission and excitation filters, allowing placing the most suitable emission filter for selecting the spectral region of interest. The output of the PTMs is expressed in photo-counts, and corresponds to the entire signal integrated in the transmission spectral band of the emission filter. Therefore, the signal acquired from a sample can also include a background contribution due to the solvent. In

important than in the case of smaller capillaries (Simeon et al., 2001).

**4.3 High-Performance Liquid Chromatography (HPLC) with LIF** 

section 3.

and dead volumes (Simeon et al., 2001).

**4.4 Photomultipliers (PTM)** 

mobile phase passes through a detection window in the LIF detector. Thus, the whole fluorescence induced by the laser is collected by the detector (Alcaide-Molina et al., 2009). In LIF detection, the number of molecules that are photo-degraded is inversely proportional to the velocity of the fluorophore in front of the laser beam (Simeon et al., 2001). The scheme of a la LIF sensor is shown on Fig. 1.

It has been said that AFB1 is the most toxic and one of the less fluorescent of the aflatoxins. However, the poorest sensitivity of the method may correspond to some other AF. Sensitivity tests should be applied for different AFs to select the one with the lowest sensitivity. The system should be calibrated with the curve of such aflatoxin; thereby, a signal provided by other AF is going to be translated into a higher concentration of this AF, leading to a confirmatory analysis on the screening method. This strategy, then, eliminates false negatives (Alcaide-Molina et al., 2009).

Thus, LIF detection shows as an appropriate detection technique with applications on very low concentrations of sample with native fluorescence or that fluoresce after derivatization (Simeon et al., 2001). However, LIF detection is a technique restricted to a limited number of laboratories because the high cost of the lasers, and because most of the analyte molecules have to be labelled with dyes that match the laser wavelength. Moreover, when the labelling reactions are not well understood, they can lead to contradictory results (Lalljie & Sandra, 1995).

Fig. 1. Scheme of a LIF detector (adapted from Simeon et al., 2001)

mobile phase passes through a detection window in the LIF detector. Thus, the whole fluorescence induced by the laser is collected by the detector (Alcaide-Molina et al., 2009). In LIF detection, the number of molecules that are photo-degraded is inversely proportional to the velocity of the fluorophore in front of the laser beam (Simeon et al., 2001). The scheme of

It has been said that AFB1 is the most toxic and one of the less fluorescent of the aflatoxins. However, the poorest sensitivity of the method may correspond to some other AF. Sensitivity tests should be applied for different AFs to select the one with the lowest sensitivity. The system should be calibrated with the curve of such aflatoxin; thereby, a signal provided by other AF is going to be translated into a higher concentration of this AF, leading to a confirmatory analysis on the screening method. This strategy, then, eliminates

Thus, LIF detection shows as an appropriate detection technique with applications on very low concentrations of sample with native fluorescence or that fluoresce after derivatization (Simeon et al., 2001). However, LIF detection is a technique restricted to a limited number of laboratories because the high cost of the lasers, and because most of the analyte molecules have to be labelled with dyes that match the laser wavelength. Moreover, when the labelling reactions are not well understood, they can lead to contradictory results (Lalljie & Sandra,

a la LIF sensor is shown on Fig. 1.

1995).

false negatives (Alcaide-Molina et al., 2009).

Fig. 1. Scheme of a LIF detector (adapted from Simeon et al., 2001)

#### **4.3 High-Performance Liquid Chromatography (HPLC) with LIF**

Fluorescence detection and electrochemical detection are the two sensitive detection means most commonly used for quantitative studies in HPLC. This happens because the sensitivity levels of those hybrid techniques are much better than the ones observed with conventional fluorescence. It has been demonstrated the usefulness of LIF for sensitive detection in HPLC and micro high-performance liquid chromatography (µHPLC) in sensing very low concentrations of substances that can be excited in the near-UV range (325 nm) after labelling at nanomolar concentrations (Folestad et al., 1985; Diebold et al., 1979). Thus, LIF-HPLC method has become very popular and an essential detection technique in capillary electrophoresis (CE). Its sensitivity has been increased by the use of photoactivation devices (Reif & Metzger, 1995). Its popularity is due to its capability to detect substances at lower ranges than the micromolar (Bayle et al., 2004). For more information about HPLC refer to section 3.

It has been said that in LIF detection, the number of molecules that are photo-degraded is inversely proportional to the velocity of the fluorophore in front of the laser beam. On the other side, the sensitivity of detection in HPLC depends on the inner diameter of the capillary connected to the output of the column. Therefore, at a constant flow-rate, the sensitivity depends on the velocity of the fluorophore in front of the laser beam of the LIF, and the solid angle of fluorescence collection by the optical arrangement (Simeon et al., 1999). As a result, the union of LIF and HPLC offers a good compromise between sensitivity and dead volumes (Simeon et al., 2001).

In flow injection experiments with LIF-HPLC systems, at a given diameter, the detector signal will increase when increasing flow-rates if photochemical degradation is a limiting factor (Simeon et al., 2001). Conversely, if the flow-rate is fixed, an increase in diameter is expected to lead to a quadratic increase in the detector volume, generating also a quadratic increase in the number of detectable molecules. Then can be said that if a larger volume is irradiated at a larger capillary diameter, the efficiency of fluorescence collection is less important than in the case of smaller capillaries (Simeon et al., 2001).

#### **4.4 Photomultipliers (PTM)**

Since Fluorescence systems have a wide sensitivity, they are a useful tool to measure AFM1 in milk, which legal limit is very low (about 50 ppt). These systems are suitable for preliminary screening at the earlier stages of the industrial process, and make it possible to discard contaminated milk stocks before their inclusion in the production chain (Cucci et al., 2007). PMTs are highly sensitive photomultipliers based flow through detection system suited for ultra low fluorescence, chemiluminescence or bioluminescence measurements (PMT-FL, FIAlab Instruments). Their photon counting photon counting sensor has a bluegreen (280–630 nm) spectral response with a peak of quantum efficiency at 400 nm and ultra-low dark counts. The high sensitivity of these devices reaches parts per trillion, permitting measurements of extremely low fluorescence signals. These devices may work with an internal excitation lamp, a LED source or an SMA terminated fiber optic cable for use with an external lamp. They also count with removable emission and excitation filters, allowing placing the most suitable emission filter for selecting the spectral region of interest. The output of the PTMs is expressed in photo-counts, and corresponds to the entire signal integrated in the transmission spectral band of the emission filter. Therefore, the signal acquired from a sample can also include a background contribution due to the solvent. In

Methods for Detection and Quantification of Aflatoxins 119

et al. (2008) in which are detected and quantified the concentration of aflatoxins B1 and B2 in pistachio. It has certain advantages in common with the FT-NIR, and low detection limit,

To detect aflatoxins in a sample, this is evaporated and mixed with a carrier gas. Then it is entered into the Ion Mobility Spectrometer (IMS) where the mixture is ionized and passed through an electric field gradient, where ions of different substances will travel at different speeds. The study by Sheibani et al. (2008) shows that using this technique is impossible to

This technique has been underutilized for the detection of aflatoxins due to calibration requirements required against standard reference chemical processes (Tripathi & Mishra, 2009). Despite of the aforementioned limitations, this technique has some advantages, such as: fast and easy equipment operation, good accuracy, precision, performing nondestructive analyzes, prediction of chemical and physical sample from a single spectrum parameters from a single spectrum enabling several components to be determined simultaneously

It basically consists of measuring the absorbance of the sample to light whose wavelength varies in the range known as the Near Infrared (NIR). In the work of Tripathi & Mishra (2009) it is found that for the correct quantification of aflatoxins B1 in chili powder network readings were taken in the range of 6900.3 - 4998.8 cm-1 and also in the range of 4902.3 - 3999.8 cm-1, excluding the water absorption bands (5155 and 7000 cm-1). Good results were obtained with respect to chemical techniques such as High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC), although its detection range is between 15 to 500 mg / kg which is slightly high compared to these

The term biosensors refers generally to a small, portable and analytical device based on the combination of recognition biomolecules with an appropriate transducer, and able of detecting chemical or biological materials selectively and with a high sensitivity (Paddle, 1996). Its principle of detection is the specific binding of the analyte of interest to the complementary biorecongnition element immobilized on a suitable support medium. When the analyte binds the element, there happens a specific interaction which results in a change of one or more physico-chemical properties. Such properties may be: pH, electron transfer, mass, or heat transfer that are detected and can be measured by a transducer. Depending of the method of signal transduction, biosensors can be divided into different groups: electrochemical, optical, thermometric, piezo-electric or magnetic. In the case of aflatoxin detection, electrochemical and optical are the most commonly used (Velasco-Garcia & Mottram, 2002). Until 1996 only few biosensors for toxins were recorded and most of them were based on ELISA. The goal of the more recent studies is to simplify and expedite the method of detection while maintenance and improvement of sensitivity is attempted

A method that has gained popularity is the use of antibodies to clean-up samples prior to measurement by LC of HPLC. Carlson et al. (2000) present an immune-affinity fluorometric

fast response, simplicity, portability, low cost.

based on the use of multivariate calibrations.

**6.2 Fourier Transform Near Infrared (FT-NIR) spectrometry** 

quantify as low as 0.25 ng.

techniques.

**7. Biosensors** 

(Sapsford et al., 2006).

principle, the latter can contribute to the actual fluorescence of the substance under analysis with a spurious signal of intrinsic fluorescence or Raman, depending on the excitation wavelength (Cucci et al., 2007).

The use of cyclodextrin (CD) as fluorescence enhancer for aflatoxins detection is widely reported in the literature (Zhilong, G. & Zhujun, 1997; Dall'asta et al., 2003), nevertheless, an increased error bar affects measurements due to the CD scattering effects.

The signal-to-noise ratio of these fluorescence measurements strongly depends on the type of cuvette used for containing the liquid sample. The cell geometry and its constituting material give rise to different effects, such as multiple reflections and stray-light. Small sample volumes and darkened walls are mandatory to achieve a better signal-to-noise ratio. Plastic cuvettes without the use of an additional fluorescence enhancer are not useful for the implementation of an early-warning system. Conversely, quartz cuvettes perform very well (Cucci et al., 2007).

Then, PTMs are compact and easy-to-handle sensors for the rapid detection of low concentrations of AFM1 in liquid solutions without the need for pre-concentration of the sample. They can be used as quick "threshold indications" and as an "early warning system", so as to rapidly single out risk/alarm situations (Cucci et al., 2007).

### **5. Ultra violet absorption**

It has been said that all the aflatoxins have a maximum absorption around 360 nm with a molar absorptivity of about 20,000 cm2 /mol (Akbas & Ozdemir, 2006). But, even though aflatoxins could be detected by UV absorption, the sensitivity of such systems is not sufficient to detect these compounds at the parts per billion (ppb) levels required for food analyses (Alcaide-Molina et al., 2009). The detection limit of UV sensors reaches micromolar ranges (Couderc et al., 1998). This is why fluorescence (FL) techniques have become more popular for AFs detection.

For overcoming the named limitation, UV absorption technique is usually combined with HPLC systems. Experimental results indicate that the detection limit of aflatoxins is enhanced by the proper method of extraction and clean-up process (Göbel & Lusky, 2004; Ali et al., 2005). For example, the selected clean-up and extraction procedures should minimize the interfering substances and matrix effect on the elution and separation of aflatoxins (Akiyama et al., 2001). Such important factors, correctly applied, may be of great importance to help the less sophisticated laboratories with HPLC instruments equipped with UV detector to detect aflatoxins with a precision that complies with the international guidelines and regulations.

Then, even though, HPLC-UV systems still are less sensitive than HPLC-FL systems, especially at low AF levels (Herzallah, 2009), HPLC-UV systems indicate to be accurate, precise, and consequently, reliable enough for determination of aflatoxins in food, with low duration and running cost.

#### **6. Spectrometry**

#### **6.1 Ion mobility spectrometry**

The Ion mobility spectrometry is a technique that is used in the characterization of chemicals on the basis of speed acquired by the gas-phase ions in an electric field. This technique has been used to determine the concentration of aflatoxins, as evidenced by the work of Sheibani

principle, the latter can contribute to the actual fluorescence of the substance under analysis with a spurious signal of intrinsic fluorescence or Raman, depending on the excitation

The use of cyclodextrin (CD) as fluorescence enhancer for aflatoxins detection is widely reported in the literature (Zhilong, G. & Zhujun, 1997; Dall'asta et al., 2003), nevertheless, an

The signal-to-noise ratio of these fluorescence measurements strongly depends on the type of cuvette used for containing the liquid sample. The cell geometry and its constituting material give rise to different effects, such as multiple reflections and stray-light. Small sample volumes and darkened walls are mandatory to achieve a better signal-to-noise ratio. Plastic cuvettes without the use of an additional fluorescence enhancer are not useful for the implementation of an early-warning system. Conversely, quartz cuvettes perform very well

Then, PTMs are compact and easy-to-handle sensors for the rapid detection of low concentrations of AFM1 in liquid solutions without the need for pre-concentration of the sample. They can be used as quick "threshold indications" and as an "early warning

It has been said that all the aflatoxins have a maximum absorption around 360 nm with a molar absorptivity of about 20,000 cm2 /mol (Akbas & Ozdemir, 2006). But, even though aflatoxins could be detected by UV absorption, the sensitivity of such systems is not sufficient to detect these compounds at the parts per billion (ppb) levels required for food analyses (Alcaide-Molina et al., 2009). The detection limit of UV sensors reaches micromolar ranges (Couderc et al., 1998). This is why fluorescence (FL) techniques have become more

For overcoming the named limitation, UV absorption technique is usually combined with HPLC systems. Experimental results indicate that the detection limit of aflatoxins is enhanced by the proper method of extraction and clean-up process (Göbel & Lusky, 2004; Ali et al., 2005). For example, the selected clean-up and extraction procedures should minimize the interfering substances and matrix effect on the elution and separation of aflatoxins (Akiyama et al., 2001). Such important factors, correctly applied, may be of great importance to help the less sophisticated laboratories with HPLC instruments equipped with UV detector to detect aflatoxins with a precision that complies with the international

Then, even though, HPLC-UV systems still are less sensitive than HPLC-FL systems, especially at low AF levels (Herzallah, 2009), HPLC-UV systems indicate to be accurate, precise, and consequently, reliable enough for determination of aflatoxins in food, with low

The Ion mobility spectrometry is a technique that is used in the characterization of chemicals on the basis of speed acquired by the gas-phase ions in an electric field. This technique has been used to determine the concentration of aflatoxins, as evidenced by the work of Sheibani

increased error bar affects measurements due to the CD scattering effects.

system", so as to rapidly single out risk/alarm situations (Cucci et al., 2007).

wavelength (Cucci et al., 2007).

(Cucci et al., 2007).

**5. Ultra violet absorption** 

popular for AFs detection.

guidelines and regulations.

duration and running cost.

**6.1 Ion mobility spectrometry** 

**6. Spectrometry** 

et al. (2008) in which are detected and quantified the concentration of aflatoxins B1 and B2 in pistachio. It has certain advantages in common with the FT-NIR, and low detection limit, fast response, simplicity, portability, low cost.

To detect aflatoxins in a sample, this is evaporated and mixed with a carrier gas. Then it is entered into the Ion Mobility Spectrometer (IMS) where the mixture is ionized and passed through an electric field gradient, where ions of different substances will travel at different speeds. The study by Sheibani et al. (2008) shows that using this technique is impossible to quantify as low as 0.25 ng.

#### **6.2 Fourier Transform Near Infrared (FT-NIR) spectrometry**

This technique has been underutilized for the detection of aflatoxins due to calibration requirements required against standard reference chemical processes (Tripathi & Mishra, 2009). Despite of the aforementioned limitations, this technique has some advantages, such as: fast and easy equipment operation, good accuracy, precision, performing nondestructive analyzes, prediction of chemical and physical sample from a single spectrum parameters from a single spectrum enabling several components to be determined simultaneously based on the use of multivariate calibrations.

It basically consists of measuring the absorbance of the sample to light whose wavelength varies in the range known as the Near Infrared (NIR). In the work of Tripathi & Mishra (2009) it is found that for the correct quantification of aflatoxins B1 in chili powder network readings were taken in the range of 6900.3 - 4998.8 cm-1 and also in the range of 4902.3 - 3999.8 cm-1, excluding the water absorption bands (5155 and 7000 cm-1). Good results were obtained with respect to chemical techniques such as High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC), although its detection range is between 15 to 500 mg / kg which is slightly high compared to these techniques.

### **7. Biosensors**

The term biosensors refers generally to a small, portable and analytical device based on the combination of recognition biomolecules with an appropriate transducer, and able of detecting chemical or biological materials selectively and with a high sensitivity (Paddle, 1996). Its principle of detection is the specific binding of the analyte of interest to the complementary biorecongnition element immobilized on a suitable support medium. When the analyte binds the element, there happens a specific interaction which results in a change of one or more physico-chemical properties. Such properties may be: pH, electron transfer, mass, or heat transfer that are detected and can be measured by a transducer. Depending of the method of signal transduction, biosensors can be divided into different groups: electrochemical, optical, thermometric, piezo-electric or magnetic. In the case of aflatoxin detection, electrochemical and optical are the most commonly used (Velasco-Garcia & Mottram, 2002). Until 1996 only few biosensors for toxins were recorded and most of them were based on ELISA. The goal of the more recent studies is to simplify and expedite the method of detection while maintenance and improvement of sensitivity is attempted (Sapsford et al., 2006).

A method that has gained popularity is the use of antibodies to clean-up samples prior to measurement by LC of HPLC. Carlson et al. (2000) present an immune-affinity fluorometric

Methods for Detection and Quantification of Aflatoxins 121

humankind. For doing this it is necessary to use methods that combine simplicity with

Adsorptive Stripping Voltammetry is a method based on accumulation and reduction of AFB1 and AFB2 species on the surface of hanging mercury drop electrode (HMDE). Such electrode offers both sensitivity and selectivity. The pioneers on this method applied to detection of aflatoxins are Hajian and Ensafi (2009), for more information refer to their

Voltammetry is an electro-analytical method. It obtains information about the sample by measuring a current while the potential is varied (Komorsky et al., 1992). The voltammetry used in the experiment of Hajian and Ensafi had three-electrodes containing hanging mercury drop electrode as a working electrode, a carbon rod as an auxiliary electrode and an Ag/AgCl (3.0 M KCl) reference electrode. This method was proved only on AFs B1 and B2, where both aflatoxins were found to adsorb and undergo irreversible reduction reaction

Adsorptive Stripping Voltammetry is an electrochemical method which has no or very low dependence on pH. This dependence displayed only for B1 in the pH range of 5.0 to 6.0 (Sun

As it is expected in adsorption processes, by increasing accumulation time, the peak currents for both of the aflatoxins are increased and then leveled off because of the saturation of electrode surface (Hajian & Ensafi, 2009). Therefore, an accumulation time of 60 seconds is recommended for improving sensitivity. It is also recommended to use the extraction and clean-up method for aflatoxins that was used by Garden and Strachan (2001). Such extraction and clean-up method try to obtain the highest yield of aflatoxins with the

This method uses single standard addition method by spiking 10 ng / ml of standard aflatoxin followed with general procedure for voltammetric analysis. The total determination of aflatoxins is based on the next formula reported by Hajian and Ensafi

> <sup>20</sup> *ng <sup>P</sup> Aflatoxin <sup>C</sup> ml P*

Where: P' is peak current of sample (nA), P is peak current of standard aflatoxin (after subtract from P') (nA), C is the concentration of aflatoxin spiked in the cell (ng/ ml) and 20 is a factor value after the sample weight, volume of methanol/water used in the extraction

Adsorptive Stripping Voltammetry is a suitable method for determination of total aflatoxins (B1 and B2) in food. This method has some advantages such as high sensitivity, extended linear dynamic range, simplicity and speed (Hajian & Ensafi, 2009). The reliability of this

Different techniques have tried to offer new options for screening aflatoxins. Screening consists on rapid and/or *in situ* detection. There are two main difficulties for an effective

and preparation of injection sample have been considered (Hajian & Ensafi, 2009).

method for determination of total aflatoxins is comparable to HPLC.

'

(1)

high detectability.

article.

et al., 2005).

(2009):

minimum matrix effect.

**9. Miscellaneous methods** 

**8. Adsorptive stripping voltammetry** 

at the working mercury electrode (Rodriguez et al., 2005).

biosensor where the sample was filtered through a column containing sepharosa beads to which the polyclonal aflatoxin-specific antibodies were joined. The beads with attached aflatoxins were subsequently rinsed to remove any impurities and interference. Posterior, an eluant solution was passed through the beads causing antibodies to release the bound aflatoxins. The analyte was collected and placed in a fluorometer. This system consists essentially of two subsystems a fluidics subsystem in charge of mechanical-handling and processing and an electro-optical system that add a miniature fluorometer.

Sapsfor et al. (2006) present a system to detect and quantify foodborne contaminants using an array biosensor. It is capable of measuring large pathogens such as the bacteria Campylobacter jejuni and small toxins (mycotoxins ochratoxin A, fumonisin B, aflatoxin B1 and deoxynivalenol). The system is capable of multiple detections of aflatoxins in a short time.

Aflatoxins have inhibitory effect on acetylcholinesterase (AchE) and their detection is coupled with the decrease in the activity of AchE which is measured using a choline oxidase amperometric biosensor (Nayak et al., 2009). Amperometric methods allow the detection of low aflatoxin concentration that cannot be detected by classical spectrophotometry because of the omission of the dilution step used in classical method.

Wang et al. (2009) present an implementation of Long range surface Plasmon – enhanced fluorescence spectroscopy (SPFS) in an immunoassay based biosensor for the highly sensitive detection of AFM1 in milk samples (LRSP). Here fluoropore-labeled molecules captured on the sensor are exited with surface plasmons (SPs) and the emitted fluorescence light is measured. The system takes the advantage of the electromagnetic intensity improvement occurring upon the resonance excitation of SPs that increase the intensity of fluorescence signal. This technique is based on surface Plasmon resonance which is becoming popular for the detection of chemical and biological species.

Others tendencies are the use of nanotechnology to detect aflatoxins such as the paper presented by Xiulan et al. (2004) where colloidal gold particles and antibodies were combined and used to develop an immunochromatographic (IC) method for aflatoxin B1 analysis. The result of this was that the analysis could be completed in less than 10 minutes and the lower test limit was 2.5ng/ml for aflatoxin B1. Such limit was increased in two times of ELISA.

When aflatoxins are consumed by cattle, they are transformed into their hydroxylated product, AFM1 that is known for its hepatotoxic and carcinogenic effects. To date, aflatoxins are regulated in many countries because of the milk intake in infants is high and when they are young the vulnerability to toxins is higher. Because of this, it is necessary to monitor AFM1 in milk at ultra low level, so that, analytical methods with high detectability and analytical throughput are required. Kanungo et al. (2011) present a novel approach where a highly sensitive microplate sandwich ELISA was developed and integrated with Magnetic nanoparticles (MNPs) which could detect ultra trace amount of AFM1 in milk. Sandwichtype immunoassay is an effective bioassay due to the high specificity and sensitivity. MNPs were used as an affinity capture column wherein immobilized antibodies on their surface could capture AFM1 from milk sample.

According to the aforementioned, the new trends could be the use of nanoparticles in combinations of the commonly used techniques such as LC, HPLC, TLC and immunoassay techniques. These combinations are to improve the detection at ultra low level of compounds in order to diminish the risk that this kind of mycotoxins causes to

biosensor where the sample was filtered through a column containing sepharosa beads to which the polyclonal aflatoxin-specific antibodies were joined. The beads with attached aflatoxins were subsequently rinsed to remove any impurities and interference. Posterior, an eluant solution was passed through the beads causing antibodies to release the bound aflatoxins. The analyte was collected and placed in a fluorometer. This system consists essentially of two subsystems a fluidics subsystem in charge of mechanical-handling and

Sapsfor et al. (2006) present a system to detect and quantify foodborne contaminants using an array biosensor. It is capable of measuring large pathogens such as the bacteria Campylobacter jejuni and small toxins (mycotoxins ochratoxin A, fumonisin B, aflatoxin B1 and deoxynivalenol). The system is capable of multiple detections of aflatoxins in a short

Aflatoxins have inhibitory effect on acetylcholinesterase (AchE) and their detection is coupled with the decrease in the activity of AchE which is measured using a choline oxidase amperometric biosensor (Nayak et al., 2009). Amperometric methods allow the detection of low aflatoxin concentration that cannot be detected by classical spectrophotometry because

Wang et al. (2009) present an implementation of Long range surface Plasmon – enhanced fluorescence spectroscopy (SPFS) in an immunoassay based biosensor for the highly sensitive detection of AFM1 in milk samples (LRSP). Here fluoropore-labeled molecules captured on the sensor are exited with surface plasmons (SPs) and the emitted fluorescence light is measured. The system takes the advantage of the electromagnetic intensity improvement occurring upon the resonance excitation of SPs that increase the intensity of fluorescence signal. This technique is based on surface Plasmon resonance which is

Others tendencies are the use of nanotechnology to detect aflatoxins such as the paper presented by Xiulan et al. (2004) where colloidal gold particles and antibodies were combined and used to develop an immunochromatographic (IC) method for aflatoxin B1 analysis. The result of this was that the analysis could be completed in less than 10 minutes and the lower test limit was 2.5ng/ml for aflatoxin B1. Such limit was increased in two times

When aflatoxins are consumed by cattle, they are transformed into their hydroxylated product, AFM1 that is known for its hepatotoxic and carcinogenic effects. To date, aflatoxins are regulated in many countries because of the milk intake in infants is high and when they are young the vulnerability to toxins is higher. Because of this, it is necessary to monitor AFM1 in milk at ultra low level, so that, analytical methods with high detectability and analytical throughput are required. Kanungo et al. (2011) present a novel approach where a highly sensitive microplate sandwich ELISA was developed and integrated with Magnetic nanoparticles (MNPs) which could detect ultra trace amount of AFM1 in milk. Sandwichtype immunoassay is an effective bioassay due to the high specificity and sensitivity. MNPs were used as an affinity capture column wherein immobilized antibodies on their surface

According to the aforementioned, the new trends could be the use of nanoparticles in combinations of the commonly used techniques such as LC, HPLC, TLC and immunoassay techniques. These combinations are to improve the detection at ultra low level of compounds in order to diminish the risk that this kind of mycotoxins causes to

processing and an electro-optical system that add a miniature fluorometer.

of the omission of the dilution step used in classical method.

becoming popular for the detection of chemical and biological species.

time.

of ELISA.

could capture AFM1 from milk sample.

humankind. For doing this it is necessary to use methods that combine simplicity with high detectability.

#### **8. Adsorptive stripping voltammetry**

Adsorptive Stripping Voltammetry is a method based on accumulation and reduction of AFB1 and AFB2 species on the surface of hanging mercury drop electrode (HMDE). Such electrode offers both sensitivity and selectivity. The pioneers on this method applied to detection of aflatoxins are Hajian and Ensafi (2009), for more information refer to their article.

Voltammetry is an electro-analytical method. It obtains information about the sample by measuring a current while the potential is varied (Komorsky et al., 1992). The voltammetry used in the experiment of Hajian and Ensafi had three-electrodes containing hanging mercury drop electrode as a working electrode, a carbon rod as an auxiliary electrode and an Ag/AgCl (3.0 M KCl) reference electrode. This method was proved only on AFs B1 and B2, where both aflatoxins were found to adsorb and undergo irreversible reduction reaction at the working mercury electrode (Rodriguez et al., 2005).

Adsorptive Stripping Voltammetry is an electrochemical method which has no or very low dependence on pH. This dependence displayed only for B1 in the pH range of 5.0 to 6.0 (Sun et al., 2005).

As it is expected in adsorption processes, by increasing accumulation time, the peak currents for both of the aflatoxins are increased and then leveled off because of the saturation of electrode surface (Hajian & Ensafi, 2009). Therefore, an accumulation time of 60 seconds is recommended for improving sensitivity. It is also recommended to use the extraction and clean-up method for aflatoxins that was used by Garden and Strachan (2001). Such extraction and clean-up method try to obtain the highest yield of aflatoxins with the minimum matrix effect.

This method uses single standard addition method by spiking 10 ng / ml of standard aflatoxin followed with general procedure for voltammetric analysis. The total determination of aflatoxins is based on the next formula reported by Hajian and Ensafi (2009):

$$\text{Aflatoxin}\left(\frac{\text{mg}}{\text{ml}}\right) = \frac{P'}{P} \ast \text{C} \ast \text{20} \tag{1}$$

Where: P' is peak current of sample (nA), P is peak current of standard aflatoxin (after subtract from P') (nA), C is the concentration of aflatoxin spiked in the cell (ng/ ml) and 20 is a factor value after the sample weight, volume of methanol/water used in the extraction and preparation of injection sample have been considered (Hajian & Ensafi, 2009).

Adsorptive Stripping Voltammetry is a suitable method for determination of total aflatoxins (B1 and B2) in food. This method has some advantages such as high sensitivity, extended linear dynamic range, simplicity and speed (Hajian & Ensafi, 2009). The reliability of this method for determination of total aflatoxins is comparable to HPLC.

#### **9. Miscellaneous methods**

Different techniques have tried to offer new options for screening aflatoxins. Screening consists on rapid and/or *in situ* detection. There are two main difficulties for an effective

Methods for Detection and Quantification of Aflatoxins 123

with optical and immunochemical techniques used to clean-up the samples achieve a better

Due to the risk that the aflatoxins represent to humans, the researchers all over the word are looking for methods to detect and quantify them. Apparently, the measurement of aflatoxins in the future tends to be the combination of optical, immunchemical, and

Authors give thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT), in Mexico, for its financial support through the scholarships with Registration Numbers: 239421 (AEC), 201401 (LMCM), 209021 (RFMH) and 207684 (JRMA). We also express our gratitude to

B-100 Series Ultra Violet Lamps, Ultra Violet Products (UVP). Nuffield Road, Cambridge,

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Akiyama, H., Goda, Y., Tanaka, T., and Toyoda, M. (2001). Determination of aflatoxins B1,

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11):1457–1472.

quantification.

fluorescence techniques.

**12. References** 

UK.

**11. Acknowledgements** 

screening method: the necessity of a very high sensitivity, which in fact is a necessity of any technique; and the demand of preliminary sample preparations. Some of these techniques, which are commented ahead, present a lack of applications because of their practical inconveniences or because they have not been proved yet with real samples (Gilbert & Vargas, 2003).

**Optical-fiber:** Modular separation based on a fiber-optic sensor (Dickens & Sepaniak, 2000) has been tested in buffers, showing enough sensitivity (0.005 ng/ml for detection of aflatoxin B1). Unfortunately, it is limited to handling only liquid matrices.

**Electrochemical transduction:** The interaction of the aflatoxin M1 with bilayer lipid membranes can be sensed electrochemically (Andreou & Nikolelis, 1997; Andreou et al., 1997) reaching a good specificity and speed of response. But, its principal negative factor is its detection limit 750 ng/ml, which is very unpractical.

**Flow injection monitoring:** Stabilized systems of filter-supported membranes are capable of achieving significantly improved sensitivity (Andreou & Nikolelis, 1998). These membranes have been proposed for use in detecting aflatoxin M1 in cheese (Siontorou et al., 2000). Single strand DNA oligomers have been incorporated into the membranes to control surface electrostatic properties. This incorporation led to achievement a sensitivity much closer to regulatory limits, and with the ability to analyze four cheese samples per minute. Even though this technique appears to be a good option for in situ testing, it does not have yet many applications (Gilbert & Vargas, 2003).

#### **10. Conclusion**

Different methods for detection and quantification of aflatoxins have been discussed along this document. Through the researching made for this document, it has been found that the most popular methods are: ELISA, electrochemical immunosensors, chromatography and fluorescence. Even though ELISA is the most common and widespread technique, it has the disadvantage of requiring well equipped laboratories, trained personnel, harmful solvents and several hours to complete an assay. The detection and quantification of aflatoxins by using electrochemical immunosensor has proven to be efficient, easy to use and able to detect very low levels of these substances. Chromatography is a method which needs immunoaffinity columns and phase solid extraction need to be used to clean-up the sample, and also pre-column and post-column derivatization to enhance the aflatoxins fluorescence properties. So that, by improving these characteristics, it is possible to obtain a better quantification and sensibility. Fluorescence detection is a very good alternative to the conventional techniques used today. It has a very high sensitivity, especially when is combined with other techniques as HPLC. Some fluorescence techniques are used even in portable sensors, resulting on in situ measurements. Techniques such as FT-NIR spectrometer and IMS have proven to be quick, inexpensive and user-friendly, however, the FT-NIR technique shows lack of sensitivity when detecting low concentrations of aflatoxins. New techniques in this field are being developed in order to give a rapid and/or in situ detection of these toxins. Some examples of these new techniques are: optical-fiber, electrochemical transduction, low injection monitoring and biosensors. All of these, except for the biosensors, still present a lack of applications because of their practical inconveniences. The biosensors have been designed to overcome the drawbacks that the common tools employed to detect and quantify aflatoxins presents. They use the inherent fluorescence property that aflatoxins have to improve the detection, that in combination with optical and immunochemical techniques used to clean-up the samples achieve a better quantification.

Due to the risk that the aflatoxins represent to humans, the researchers all over the word are looking for methods to detect and quantify them. Apparently, the measurement of aflatoxins in the future tends to be the combination of optical, immunchemical, and fluorescence techniques.

#### **11. Acknowledgements**

Authors give thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT), in Mexico, for its financial support through the scholarships with Registration Numbers: 239421 (AEC), 201401 (LMCM), 209021 (RFMH) and 207684 (JRMA). We also express our gratitude to CONACYT for funding the project CB-2008-01.000000000106133.

#### **12. References**

122 Aflatoxins – Detection, Measurement and Control

screening method: the necessity of a very high sensitivity, which in fact is a necessity of any technique; and the demand of preliminary sample preparations. Some of these techniques, which are commented ahead, present a lack of applications because of their practical inconveniences or because they have not been proved yet with real samples (Gilbert &

**Optical-fiber:** Modular separation based on a fiber-optic sensor (Dickens & Sepaniak, 2000) has been tested in buffers, showing enough sensitivity (0.005 ng/ml for detection of

**Electrochemical transduction:** The interaction of the aflatoxin M1 with bilayer lipid membranes can be sensed electrochemically (Andreou & Nikolelis, 1997; Andreou et al., 1997) reaching a good specificity and speed of response. But, its principal negative factor is

**Flow injection monitoring:** Stabilized systems of filter-supported membranes are capable of achieving significantly improved sensitivity (Andreou & Nikolelis, 1998). These membranes have been proposed for use in detecting aflatoxin M1 in cheese (Siontorou et al., 2000). Single strand DNA oligomers have been incorporated into the membranes to control surface electrostatic properties. This incorporation led to achievement a sensitivity much closer to regulatory limits, and with the ability to analyze four cheese samples per minute. Even though this technique appears to be a good option for in situ testing, it does not have yet

Different methods for detection and quantification of aflatoxins have been discussed along this document. Through the researching made for this document, it has been found that the most popular methods are: ELISA, electrochemical immunosensors, chromatography and fluorescence. Even though ELISA is the most common and widespread technique, it has the disadvantage of requiring well equipped laboratories, trained personnel, harmful solvents and several hours to complete an assay. The detection and quantification of aflatoxins by using electrochemical immunosensor has proven to be efficient, easy to use and able to detect very low levels of these substances. Chromatography is a method which needs immunoaffinity columns and phase solid extraction need to be used to clean-up the sample, and also pre-column and post-column derivatization to enhance the aflatoxins fluorescence properties. So that, by improving these characteristics, it is possible to obtain a better quantification and sensibility. Fluorescence detection is a very good alternative to the conventional techniques used today. It has a very high sensitivity, especially when is combined with other techniques as HPLC. Some fluorescence techniques are used even in portable sensors, resulting on in situ measurements. Techniques such as FT-NIR spectrometer and IMS have proven to be quick, inexpensive and user-friendly, however, the FT-NIR technique shows lack of sensitivity when detecting low concentrations of aflatoxins. New techniques in this field are being developed in order to give a rapid and/or in situ detection of these toxins. Some examples of these new techniques are: optical-fiber, electrochemical transduction, low injection monitoring and biosensors. All of these, except for the biosensors, still present a lack of applications because of their practical inconveniences. The biosensors have been designed to overcome the drawbacks that the common tools employed to detect and quantify aflatoxins presents. They use the inherent fluorescence property that aflatoxins have to improve the detection, that in combination

aflatoxin B1). Unfortunately, it is limited to handling only liquid matrices.

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**10. Conclusion** 


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**8** 

Imtiaz Hussain

*Pakistan* 

**Aflatoxin Measurement and Analysis** 

Much research work has been devoted over the last 40 years for developing methods for detection and determination of aflatoxins in foods and agriculture commodities (Chu, 1991; Holcomb, et al., 1992). This effort is continuing and keeping pace with the progress in analytical chemistry. Methods for aflatoxins are required to meet the legislation, monitoring and survey work, and for research. Different highly efficient and sophisticated techniques have been developed in the recent years for the determination of aflatoxins in different commodities. Presently the most commonly used methods for detection of aflatoxins are: high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and enzyme-linked immunosorbent assay (ELISA) (Lee et al., 2009) and fluorometeric method (Hansen, 1990). All analytical procedures include the steps: sampling, extraction, clean- up (purification) and determination (identification and quantification). The analytical detail in this chapter has been discussed in three sub-groups: sample preparation techniques,

Sampling and sample preparation is of utmost importance in the analytical identification of aflatoxins. It certainly affects the final conclusion. For the determination of aflatoxins at the parts-per-billion level, the systematic approaches to sampling, sample preparation and analysis are absolutely necessary. European Union has formed specific plans for certain commodities e.g. corn and peanuts. The performance of sampling plans for aflatoxin in granular feed products, such as shelled maize (Johansson, et al., 2000) and cotton seed (Whitaker et al., 1976) has been evaluated, while there has been little evaluation of sampling

In case of sampling of solid commodities the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample. In case of sampling of liquid commodities like milk, due to homogeneous distribution of aflatoxins in liquid milk, there is less uncertainty in aflatoxin measurement in milk. After proper sampling, there are the steps of extraction and clean-up. Sometimes extraction and clean-up is the same step and sometimes extraction is different step and clean-up is a different step. Extraction of samples, together with effective clean-up step, is an essential step in the analysis of aflatoxins. The analyte migrates into the extraction solvent. The interfering compounds are removed by clean-up step. Common extraction

detection techniques and typical complete procedures.

solvents for aflatoxins are acetonitrile-water and methanol-water.

**2. Sample preparation techniques** 

plans to detect aflatoxin in milk.

**1. Introduction** 

*Govt. Post Graduate College Samundri, Faisalabad,* 

